Most breast cancer deaths result from relapse wherein sites of latent disease escape from a dormant state. Remarkably, the latent foci of residual disease that beget breast cancer relapse remain uncharacterized despite their enormous clinical importance. Therefore, very little is known about the biology of breast cancer dormancy and escape. Since sites of latent disease in breast cancer patients are likely to remain inaccessible to researchers for the foreseeable future, modeling dormant breast cancer in genetically-modified mice offers an alternative means to: 1) uncover the biological basis for tumor dormancy, and 2) design and test rational treatment strategies aimed at eradicating dormant disease. With these goals in mind, a mouse mammary tumorigenesis model was developed in which a genetic event responsible for initiating breast cancer (activation of the Wnt signaling pathway) can be reversed. Shutting off Wnt signaling triggers regression of these cancers, but long-lived disease lesions persist and typically remain subclinical for extended periods before they escape from dormancy and beget tumor relapse. By recapitulating key clinical features of dormant breast cancer, this model provides novel experimental access to sites of latent disease, enabling elucidation of the cellular and molecular mechanisms that maintain dormancy. Moreover, the disease-free interval in this model provides a window for pre-clinical testing of treatment strategies directed against dormant cancer. In Aim1 of the proposal, conventional cytotoxic agents will be employed in pre-clinical modeling to determine how to optimally time treatment to prevent tumor escape. Here, outcomes will be compared when cytotoxic agents are administered before vs. during vs. after the initiation of dormancy. In Aim 2, genetically encoded reporters will be used to track tumor cell divisions within latent tumors to determine whether dormant mammary cancers are maintained by a subpopulation of quiescent, treatment-resistant tumor cells. These studies will test the concept that eradicating dormant cancer critically depends on targeting a quiescent subset of tumor cells that are crucial for relapse. In Aim 3, genetic pathways that drive escape from tumor dormancy will be discovered using a genetic screen that relies on the mobilization of a transposon (jumping gene) in mammary cancers. This screen is designed to identify novel therapeutic targets for relapse prevention. Ultimately, our studies will aid in developing more rational approaches for preventing breast cancer relapse by eradicating sites of dormant disease.